Bottom Line:
Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging.This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells.It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

ABSTRACTVimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

Mentions:
To generate vimentin-specific nanobodies, an alpaca (Vicugna pacos) was immunized with recombinant vimentin. Subsequently, a phagemid library (~2 × 107 clones) was established from peripheral blood mononuclear cells (PBMCs), representing the respective Nb repertoire. After two cycles of biopanning against full-length vimentin, 47 single clones were analyzed in a solid-phase phage ELISA (Supplementary Fig. 1). Sequencing of the positive clones resulted in eight unique Nb sequences (Fig. 1a). Analysis of the hallmark residues (VH/VHH: V37Y; G44Q; L45R; W47L) located in framework 2 revealed that four Nbs (VB3, VE3, VG1 and VF6) are derived from heavy-chain-only antibodies, whereas VB6, VC4, VG4 and VH3 are VH domains derived from conventional IgGs36. To identify suitable candidates for the envisaged intracellular studies, all selected binders were analyzed in living cells. To this end, we generated chromobody constructs by fusing the coding sequences of the selected Nbs to eGFP and transiently expressed them in HeLa cells. The chromobodies VE3-CB and VH3-CB formed intracellular aggregates, whereas VC4-CB, VF6-CB and VG4-CB were diffusely distributed. Notably, VB3-CB, VB6-CB and VG1-CB displayed a filamentous pattern that resembles the distribution of vimentin (Fig. 1b). Additional structures reminiscent of a midbody pattern were observed in the majority of VG1-CB expressing cells. Hence, we selected VB3 nanobody/chromobody and VB6 nanobody/chromobody as best candidates for further biochemical and cell-biological studies.

Mentions:
To generate vimentin-specific nanobodies, an alpaca (Vicugna pacos) was immunized with recombinant vimentin. Subsequently, a phagemid library (~2 × 107 clones) was established from peripheral blood mononuclear cells (PBMCs), representing the respective Nb repertoire. After two cycles of biopanning against full-length vimentin, 47 single clones were analyzed in a solid-phase phage ELISA (Supplementary Fig. 1). Sequencing of the positive clones resulted in eight unique Nb sequences (Fig. 1a). Analysis of the hallmark residues (VH/VHH: V37Y; G44Q; L45R; W47L) located in framework 2 revealed that four Nbs (VB3, VE3, VG1 and VF6) are derived from heavy-chain-only antibodies, whereas VB6, VC4, VG4 and VH3 are VH domains derived from conventional IgGs36. To identify suitable candidates for the envisaged intracellular studies, all selected binders were analyzed in living cells. To this end, we generated chromobody constructs by fusing the coding sequences of the selected Nbs to eGFP and transiently expressed them in HeLa cells. The chromobodies VE3-CB and VH3-CB formed intracellular aggregates, whereas VC4-CB, VF6-CB and VG4-CB were diffusely distributed. Notably, VB3-CB, VB6-CB and VG1-CB displayed a filamentous pattern that resembles the distribution of vimentin (Fig. 1b). Additional structures reminiscent of a midbody pattern were observed in the majority of VG1-CB expressing cells. Hence, we selected VB3 nanobody/chromobody and VB6 nanobody/chromobody as best candidates for further biochemical and cell-biological studies.

Bottom Line:
Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging.This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells.It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

ABSTRACTVimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.